Expression of transforming growth factor-β (TGF-β) in chronic idiopathic cough

Xie Shaoping, Macedo Patricia, Hew Mark, Nassenstein Christina, Lee Kang-Yun, Chung, Chung Kian

Description

In patients with chronic idiopathic cough, there is a chronic inflammatory response together with evidence of airway wall remodelling and an increase in airway epithelial nerves expressing TRPV-1. We hypothesised that these changes could result from an increase in growth factors such as TGFβ and neurotrophins. We recruited 13 patients with persistent non-asthmatic cough despite specific treatment of associated primary cause(s), or without associated primary cause, and 19 normal non-coughing volunteers without cough as controls, who underwent fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) and bronchial biopsies. There was a significant increase in the levels of TGFβ in BAL fluid, but not of nerve growth factor(NGF) and brain-derived nerve growth factor(BDNF) compared to normal volunteers. Levels of TFGβ gene and protein expression were assessed in bronchial biopsies. mRNA expression for TGFβ was observed in laser-captured airway smooth muscle and epithelial cells, and protein expression by immunohistochemistry was increased in ASM cells in chronic cough patients, associated with an increase in nuclear expression of the transcription factor, smad 2/3. Subbasement membrane thickness was significantly higher in cough patients compared to normal subjects and there was a positive correlation between TGF-β levels in BAL and basement membrane thickening. TGFβ in the airways may be important in the airway remodelling changes observed in chronic idiopathic cough patients, that could in turn lead to activation of the cough reflex.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2009
• Nombre de lectures: 9
• Langue: English

Extrait

BioMed CentralRespiratory Research
Open AccessResearch
Expression of transforming growth factor-  (TGF- ) in chronic
idiopathic cough
1 1 1 2Shaoping Xie , Patricia Macedo , Mark Hew , Christina Nassenstein ,
Kang1 1Yun Lee and Kian Fan Chung*
1Address: Airway Disease Section, National Heart & Lung Institute, Imperial College & Royal Brompton Hospital, London SW3 6LY, UK and
2Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany
Email: Shaoping Xie - [e-mail]; Patricia Macedo - [e-mail]; Mark Hew - [e-mail];
Christina Nassenstein - [e-mail]; Kang-Yun Lee - [e-mail]; Kian Fan Chung* - [e-mail]
* Corresponding author
Published: 22 May 2009 Received: 11 July 2008
Accepted: 22 May 2009
Respiratory Research 2009, 10:40 doi:10.1186/1465-9921-10-40
This article is available from: http://respiratory-research.com/content/10/1/40
© 2009 Xie et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
In patients with chronic idiopathic cough, there is a chronic inflammatory response together with
evidence of airway wall remodelling and an increase in airway epithelial nerves expressing TRPV-1.
We hypothesised that these changes could result from an increase in growth factors such as TGF 
and neurotrophins.
We recruited 13 patients with persistent non-asthmatic cough despite specific treatment of
associated primary cause(s), or without associated primary cause, and 19 normal non-coughing
volunteers without cough as controls, who underwent fiberoptic bronchoscopy with
bronchoalveolar lavage (BAL) and bronchial biopsies.
There was a significant increase in the levels of TGF  in BAL fluid, but not of nerve growth
factor(NGF) and brain-derived nerve growth factor(BDNF) compared to normal volunteers. Levels
of TFG  gene and protein expression were assessed in bronchial biopsies. mRNA expression for
TGF  was observed in laser-captured airway smooth muscle and epithelial cells, and protein
expression by immunohistochemistry was increased in ASM cells in chronic cough patients,
associated with an increase in nuclear expression of the transcription factor, smad 2/3.
Subbasement membrane thickness was significantly higher in cough patients compared to normal
subjects and there was a positive correlation between TGF-  levels in BAL and basement
membrane thickening.
TGF  in the airways may be important in the airway remodelling changes observed in chronic
idiopathic cough patients, that could in turn lead to activation of the cough reflex.
Background patients, no cause can be identified despite thorough
Chronic cough is a common clinical problem [1,2]. investigations and empiric treatment [4-6], a group
Asthma, postnasal drip or rhino-sinusitis, and gastro- recently denoted as 'idiopathic'. Patients with chronic
oesophageal reflux have been recognized as being the cough very often demonstrate an increased tussive
most common causes of chronic cough [2,3]. In some response to inhalation of tussive agents such as capsaicin
Page 1 of 10
(page number not for citation purposes)Respiratory Research 2009, 10:40 http://respiratory-research.com/content/10/1/40
Table 1: Patient characteristicsindicates that there is a sensitisation of the cough reflex
[7]. Both peripheral and central causes of this
sensitisaNormals Chronic cough
tion have been put forward[8,9]; however, changes
observed in the airways of patients with chronic cough
Number 13 20indicate that peripheral changes could be involved in the
sensitisation of the cough reflex. Thus, there is an increase
in mediator expression as measured by increased levels of Gender (Male/Female) 9:4 4:16**
histamine in bronchoalveolar lavage fluid, and levels of
cys-leukotrienes, leukotriene B4, myeloperoxidase and
Age (years) 19.9 ± 0.4 55.4 ± 2.5**
TNF  in induced sputum samples from patients with
persistent cough [10]. Examination of bronchial biopsies
Smoking status (n)from non-asthmatic chronic cough patients reveal an
increase in mast cells in the submucosa, with also marked
changes in airway wall remodelling such as subepithelial never 12 11
fibrosis, goblet cell hyperplasia and blood vessels, similar
to that observed in patients with asthma, together with an
ex-smoker 1 9
increase in airway smooth muscle cells [11]. Perhaps of
greater relevance to the enhanced cough reflex are
abnorPack-years 7 13.1 ± 3.5malities in the epithelial nerve profiles which could
represent cough receptors. Although there are no increases in
nerve profiles, the expression of the neuropeptide, calci- GORD (n) NA 10
tonin gene-related peptide (CGRP), and of the ion chan- Postnasal drip (n) NA 7
Neither (n) NA 5nel, transient receptor potential vanniloid 1 (TRPV1), has
been reported to be increased in these epithelial nerves
[12,13]. Atopy (%) 31 26
To explore further the role of airway wall remodelling and
Capsaicin (log C5) ND 0.53 ± 0.1410 of peripheral neural plasticity in chronic cough, we have
measured in bronchoalveolar lavage fluid the levels of
growth factors, such as transforming growth factor-  FEV (% predicted) 96.0 ± 3.3 99.0 ± 2.31
(TGF ), which may be involved in subepithelial fibrosis
[14], and of the neurotrophins such as brain-derived
neuFVC (% predicted) 100.5 ± 3.3 96.8 ± 4.6
rotrophin (BDNF) which may elicit sensitisation of
nociceptors [15], and angiogenesis and microvascular
Bronchoalveolar lavageremodelling [16]. We also examined the expression of
TGF  in airways submucosa of chronic idiopathic cough
patients. % macrophages 97.6 ± 0.5 90.1 ± 2.6**
Methods % neutrophils 1.5 ± 0.4 6.7 ± 2.6*
Subjects
We studied patients with chronic cough of at least 8
% lymphocytes 0.7 ± 0.2 3.0 ± 0.8**weeks' duration referred to our cough clinic and excluded
patients who had a diagnosis of asthma as a cause of their
cough (Table 1). As a control group, we recruited normal % eosinophils 0.3 ± 0.1 0.2 ± 0.1
volunteers through local advertisement; these normal
volunteers had no previous history of cough or asthma, and
FEV : Forced expiratory volume in one second; FVC: Forced vital 1
were not suffering from any intercurrent illness. capacity; GORD: gastro-oesophageal reflux disease; NA: Not
applicable; ND: Not done. Data shown as mean ± SEM. * p < 0.05; **p
< 0.01.Patients with chronic cough underwent diagnostic
evaluation that included chest radiograph, pulmonary function
[PC - FEV ] < 4 mg/ml), diurnal variation of peak expir-20 1test, methacholine challenge, 24-hour oesophageal
pHatory flow (> 20%), or > 15% increase of FEV after -ago-1 monitoring, and chest and sinus computed
tomogranist, and also response of coughing to inhaled
phy[1]. Patients with airway hyperresponsiveness
(probronchodilator and corticosteroid therapy were
diagvocative concentration of methacholine that induced a >
nosed as having asthma responsible for chronic cough,
20% decrease of forced expiratory flow in 1 second (FEV )1 and they were excluded from the study. The patients with
Page 2 of 10
(page number not for citation purposes)Respiratory Research 2009, 10:40 http://respiratory-research.com/content/10/1/40
chronic cough recruited to this study had a PC FEV > 8 For TGF- 1 assay, BALFs were first activated by incubation20 1
mg/ml. Chronic cough due to gastro-oesophageal reflux with 1N HCl for 10 min and neutralized by 1.2 N NaOH/
was diagnosed by 24-hour oesophageal pH-monitoring 0.5 M N-2-hydroxyethylpiperazine-N'-ethane sulfonic
and efficacy of 12-week course of proton-pump inhibitor, acid. Activated samples were then transferred to the wells
and dietary changes. Chronic cough was attributed to of plates coated with TGF- 1 soluble receptor Type II. For
post-nasal drip/rhinosinusitis when symptoms and objec- NGF and BDNF assay, plates were coated with
antitive diagnosis of postnasal drip and/or rhinosinusitis were human -NGF or BDNF antibody. 100 l of BALFs were
present and nasal corticosteroids and/or nasal anticholin- added to each well. After incubation and thorough
washergics were effective against cough. Some patients had no ing, specific antibody for each measurement was added to
identifiable cause(s) of cough despite additional investi- the test wells. TGF- 1, NGF and BDNF were detected
gations including bronchoscopy and intensive therapeutic using a horseradish peroxidase-based colorimetric assay.
trials for asthma, gastro-esophageal reflux and postnasal
drip/rhinosinusitis, and were labelled as 'idiopathic'. Laser Capture Microdissection
Only ex-smokers who have ceased smoking more than 12 Human airway biopsies were embedded in Optimum
months of enrolled were recruited. Cutting Temperature compound (OCT) on dry ice and
snap-frozen in liquid nitrogen before storage at -80°C.
The study was approved by the Ethics Committee of our Frozen sections were cut at 6 m thickness and mounted
institution and all subjects gave informed consent to par- on Laser Capture